PACIFIC SOUTHWEST ASSOCIATION OF CHEMISTRY TEACHERS
CHEMISTRY OF CITRUS FRUITS'
0
GLENN H. JOSEPH Sunkist Growers Research, Corona, California
flAVEDO or iPlCARP
A DISCUS~ION of
citrus chemistry 35 years ago would have been relatively simple. The American citrus products industry a t that time was an infant. Citric acid was the principal chemical substance being produced, although interest was keen in attempts to produce oils of oratlge and lemon of sufficient quality and at the proper price so as to compete with the well-accepted, imported oils from Southern Europe. The total citrus production in the United States jumped from 56,000,000 packed boxes in 1926 to 196,000,000 in 1946. Since 1946 large acreages have been planted in some regions while nearly similar acreaees have been removed in other areas, so the total annual production of all varieties of citrus in this country remains at about 200,000,000 packed boxes. This astounding growth of the industry has led to demands that chemists learn more and more about the fruits so &s to expand the list of saleable products. The complexity of the chemistry of citrus has resulted in the development of specialists in narrow fields of citrus research. No longer are there experts in the whole general realm of citrus chemistry. This discussion will endeavor to show the structures and characteristic reactions of typical compounds of citrus fruits, illustrating the diverse fields of chemistry which are involved with commercial products. No attempt will be made to discuss the biogenesis of the several classes of compounds. We shall start with fully matnre fruit8 and examine the principal chemical classes we meet as we go from the rind to the seeds (see tigure). THE FLAVEDO (EPICARP)
Their bright colors and exotic fragrance have made citrus fruits items noted by poets, philosophers, and historians over the past several thousand years. The compounds mainly responsible for these two characteristics are the carotenoid pigments and the essential oils, two types of compounds located in the flavedo of the fruit. Carotene (C40H6B), a yellow pigment, and xanthophyll (C40H6602) are responsible for the yellow to orange and red colors of citrus fruits. These two caro-
' Prepared for presentation at a Symposium on "Chemistry in the Citrus Fruit Industry," sponsored by the Divisions of Industrial and Engineering Chemistry and Agricultural and Food Chemistry at the 131st Meeting of the American Chemical Society, Miami, April, 1957. 'VOLUME 34, NO.
10,
OCTOBER, 1951
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Considerable commercial importance is attached to compounds n.hich are found in oil glands scattered in the flavedo or epicarp, adjacent to the chromoplasts. These tiny oil glands house the valuable essential oils of citrus, mixtures of terpenes, sesqoiterpenes, alcohols, aldehydes, ketones, esters, ethers, sterols, and many nt.her rhemiral tvnes. One of the most olentiful com..--.- ~ - - ~~~- ~ ~" nonents of citrus essential oils is the cyclic hydrocarbon, hlimonene. This terpene hydrocarbon (C,H2,-,) appears to have been made by the union of two isoprene molecules. Limonene is AIWO)-menthadiene. The terpenes have
Isoprene (2 moles)
Iimonene
the formula ClaH16, hence C6H8 compounds are known as hemiterpenes (isoprene is an example), and those with the formula (CJ& are known as sesquiterpenes. An example of this latter class is bisabolene, a constituent of citrus oils. Many alcohols are present in the essential oils from citrus, such as the unsaturated alcohols, geraniol and its sterioisomer nerol, (CIOHIPO).One aldehyde, citral, is quite important in lemon oil. This straight-chain, unsaturated aldehyde cyclizes easily to form a terpenelike compound. Citral is believed by many t o be a starting point for the formation of a vast array of cyclic
Citral
I
I
\ CHO I1
\ CHSOH I
A
Lirnonene
II
A
Gemniol
terpenes. There are several standard methods for determining citral, since the content of citral in lemon oil is of considerableinterest in assaying that oil. The wellknown phenylhydrazine reaction for aldehydes constitutes the basis for one of the most used assay procedures. The flavedo also contains, in the oils, many sterols, actually phytosterols, since they are of plant origin. Berzelius designated the part which crystallized upon standing as "stearoptene." An example of this type of compound is citroptene, C,,HloOn, which is really a derivative of conmarin. These compounds must be removed from the citrus oils if alcoholic extracts of great permanent clarity are desired. THE ALBEDO (MESOCARP)
The albedo, or white layer immediately under the outer, colored rind of citrus fruits, is usually about onethird pectic substances and one-third cellulosic material, on the dry basis. The remainder is smaller percentages of flavonoids, polysaccharides and monosaccharides such as glucose and fructose. Cellulose has received the widest attention and is perhaps better known than are the pectic substances or the recently publicized flavonoids. Cellulose structures and reactions will be omitted from this discussion. Only some characteristic formulas and analytical reactions for pectic and flavonoid subsances will be presented. Pectic Substances. I t is proper perhaps to discuss the pectic suhstances under the heading of the albedo or mesocarp because it is in this part of citrus fruits that pectic suhstances are most abundant. Actually these colloidal materials are present in all cellular structures of the fruit and play a most important part, along with cellulose, in giving form and mechanical strength. It surely cannot be just accidental that pectic substances and the cellulosic materials are together in the
carpellary membranes and vesicles and that they have such tremendous water holding capacities. Consider for a moment the fact that in an orange the segment covers and juice sacs have a moisture content of about. 88% (12% nonaqueous material) and that the juice contains about 12y0 nonaqueous substances (about 88% water). Here then are two systems, each containing about 88% water, yet one of them, the structural framework, is firm enough to protect the edible portion during its life on the tree and while it is being buffeted by picking, washing, packing, and shipping. The fruit on the tree must also withstand the rigors of cold, tropical heat, and fierce winds, yet the wonderful strength and form-giving frame Nature put in the edible portion contains no more nonaqueous chemical substances than does the juice within the fruits. What chemist could devise a better system and who among us is without awe for the Master Chemist who worked out the intricacies of these marvels of Nature's handiwork? Pectic substances are polysaccharides which contain uronic acids. They are polyuronides, in particular, polygalacturonides. This distinguishes them from the polymannuronides of sea plants and the aldobionic acids (complex group of galactose and glucuronic acid units) of desert bushes and trees. Their aldehyde endgroups are involved with linkages among the units while the carboxyl groups are free, methoxylated, or combined with alkali or alkaline-earth metals. Pectin associated naturally with hemicelluloses and cellulose in a water insoluble form is termed "protopectin." It is the precursor of the well-known watersoluble pectin of commerce. Protopectin is easily converted to water-soluble pectin by the aid of heat, acids, or certain enzymes. It should be possible, theoretically, t o find a methoxyl content of 16.32% in pectin. Usually the methoxyl content for pectins of commerce is in the range bf 8-ll%, while, for certain uses, it has been found desirable to reduce the methoxyl to the 3.55.0% region where the remaining molecule exhibits reactions with metallic ions typical of normal ionic bonding. I n certain commercial pectins one finds part of the carboxyl groups combined as methyl esters, others as salts of alkali or alkaline-earth metals, and some as ammonium salts. I n certain partially de-esterified pectins some carboxyls are converted to acid amides. The chemical determination of commercial pectin quality or quantity can involve direct titration for the free carboxyls, titration after adding formaldehyde to get the ammonium salts, ashing and determining the metal content of the ash, and saponifying to learn the ester content. Distillation from sodium hydroxide solution reveals the combined ammonium and acid amide content. Many colorimetric methods are available, mainly those where some hydrolytic product of pectin produces a specific color. When pectic suhstances are distilled at 120°C. with 12% HCI, the galacturouic acid loses C02 and forms the pentose, arabinose, which on further heating is quantitatively converted to furfural by losing three molecules of HzO. The COnis determined by absorption in B ~ ( O H )while Z furfural is measured by a colorimeter after reaction with aniline. Intricate physical chemistry is involved when one determines average molecular weights of pectins. One scheme is to nitrate the pectin (with very high density
AOOH d.Galacturonic acid
ments even though those found in citrus are usually colorless when pure. These compounds may all be considered as derivatives of y-pyrone, a ring of 5 carbons and 1 oxygen, with a quinoid oxygen on the carbon para to the oxygen of the ring. The simplest aromatic derivative is benzopyrone with a plain benzene ring having one side in common with the y-pyrone ring. A phenyl group attached to the carbon adjacent t o the oxygen in the pyrone ring gives the real basic unit for the flavone derivatives, 2-phenyl-benzopyrone. The flavonoids are thus derivatives of a C e C s C s skeleton. Sugars, usually both rhamnose and glucose, are attached to carbon 7 for citrus flavonoids, with only a few cases where rhamnose alone is attached to carbon 7. When sugars are attached to the flavonoid nucleus, the compounds are called glycosides and when the sugars have been removed or are not present the term aglycme is applied. When flavonoids are treated with alkali they are converted into the soluble chalcone form. The oxonium salt or a flavylium-like salt is formed (usually the chloride) by reduction in alcoholic hydrochloric acid. The flavylium-like salts exhibit colors ranging from red to magenta, characteristic of the compounds used, and form the basis for a colorimetric assay. The chalcone form lends itself nicely t o colorimetric assay due to the unique development of a yellow color when allowed to react in anhydrous acetone with boric and citric acids. (See structures below.)
(pentose)
acid distillation breaks oxygen linkage
H OH ..'OH
/ ..
:
HCH : H
